3.1. Adaptive Immune Response in CBM

One of the first published works in the literature observed that murine models of CBM showed disseminated disease, with live fungi in organs like the brain, liver, lungs, heart, spleen, and kidneys. The authors suggested that the cellular response is more protective to the host than the humoral response [70]. The significance of T lymphocytes in the control of CBM disease has been observed in experiments using nude animals (T-cell deficient mice). In the absence of T-cells, a severe disease was observed in these animals. However, after an adoptive T-cell transfer, the infected animals showed complete remission from the disease [71]. The following studies demonstrated that the CD4⁺ T lymphocytes are the subpopulation responsible for the control of the disease. The absence of CD8⁺ T lymphocytes did not interfere with the severity of the disease, at least in murine CBM [72]. Since CD4⁺ lymphocytes (especially Th1 subset) are important to IFN-γ production, the role of CD4⁺ lymphocytes was also observed in humans, whereby patients with mild symptoms of CBM showed higher levels of IFN-γ and lower levels of IL-10 than patients with severe symptoms of CBM [73,74]. Recently, in vitro experiments showed that F. pedrosoi drives CD4⁺ Th17 cell differentiation [32,75], while an increase in the IL-17 level was observed in skin lesions of murine CBM [76]. IL17 was also showed to be important in the early stage of the disease. Animals that had IL-17 blocked by anti-IL-17 injections showed a greater fungal burden during the early stages of the disease [77]. IL-17 is an important cytokine that is related to AMP release in the skin and fungal infections [78,79,80], helping with the killing of extracellular pathogens, such as hyphae. Although related to the polarization of anti-inflammatory macrophages (M2 macrophages) [81,82], IL-17 is an important cytokine for neutrophil migration. It is related to the increases in MIP-2 and KC, important chemokines in neutrophil attraction [83,84]. Therefore, the blockage of IL-17 could interfere with AMP release and neutrophil attraction to the infection site, leading to an increase in the fungal burden; thus, neutrophils are important in F. pedrosoi conidia and hyphae killing (as discussed below). However, the roles of the Th17 cell population and IL-17 in the control of CBM disease still remain unclear.

Although the cellular response is important for CBM control, the role of the humoral response in the disease is not fully determined yet. In the 1980s, one group demonstrated the capacity of F. pedrosoi-specific IgG antibodies to decrease 50% to 60% of fungal growth in vitro [85]. Later, a specific antibody to F. pedrosoi melanin was isolated and demonstrated to inhibit fungal growth and increase human neutrophil phagocytosis, as well as reactive oxygen species (ROS) production [86]. In 2010, Machado and colleagues observed that B-cell-deficient mice (Xid animals) had more severe CBM than wild-type animals, suggesting that the humoral response is essential for the control of the disease [87]. However, the patients’ samples showed conflicting results about the importance of the humoral response in CBM disease control. Esterre and colleagues first observed that IgG levels were directly related to disease severity [88], but this finding was not observed by Gimenez and colleagues [73]. Therefore, the actual consensus is that the CD4⁺ Th1 lymphocyte response is associated with better control of the disease, while the CD4⁺ Th2 lymphocytes are related to poor control and higher disease severity [89] (Figure 1). The importance of a humoral response in CBM disease needs deeper and specific studies.

Figure 1. PRRs and immune response in chromoblastomycosis (CBM) infection: Membrane receptors of the TLR and CLR families described in F. pedrosoi conidia recognition. To date, only one receptor is described in hyphae recognition—the intracellular NLRP-3 inflammasome—and none is described in muriform cell recognition (Bottom). The Th1 and Th17 response is associated with mild disease, and an increase in IFN-γ and IL-17 secretion, while a Th2 response is related to severe disease with increases in IL-4 and IL-10 secretion and fungal spread (Top).

3.2. Innate Immune Response in CBM

Most studies of CBM’s immune response have focused on the innate immune response. In 1984, Torinuki and colleagues observed the activation of the alternative pathway of the complement system over F. pedrosoi [90]. Thereafter, it was identified that melanin, present in a high concentration in the F. pedrosoi cell wall, was triggering the activation of the alternative pathways of the complement system [91,92]. The complement system’s activation seems to be important for the opsonization (C3b) and chemotaxis process (C3a and C5a), which drives the innate immune response towards fungal clearance. However, due to the F. pedrosoi cell wall thickness, it is highly unlikely that the complement system acts to directly eliminate F. pedrosoi through formation of the membrane attack complex. Another important innate immune component is the macrophage. In vitro experiments have demonstrated that F. pedrosoi conidia ingested by peritoneal resident macrophages can survive and grow inside the macrophages, transforming into hyphae particles and leading to macrophage death through mechanical disruption of the cytoplasmic membrane [42]. Previous activation with IFN-γ will enable macrophages to block fungal growth inside it, acting as a Trojan horse and carrying live fungal particles around the host [93]. The inability of macrophages to kill ingested conidia is due to the capacity of F. pedrosoi to block nitric oxide production in macrophages, even after IFN-γ activation [94,95]. Therefore, although the levels of IFN-γ are related to a mild form of the disease, the mechanism of IFN-γ to act against CBM agents is still unclear. Unlike macrophages, neutrophils show a high in vitro microbicidal activity against F. pedrosoi [47,96]. In 2011, in an analysis of skin biopsies from mice infected with three different forms of F. pedrosoi (conidia, sclerotic bodies, and hyphae), Machado and colleagues observed dead fragments or injured fungal particles in neutrophilic areas, while intact or particles similar to the live particles were observed in regions of macrophage cells. These findings suggest that neutrophils are important for F. pedrosoi killing, not only in vitro but also in vivo. The authors also suggested that neutrophils release Neutrophil Extracellular Traps (NETs) to kill hyphae particles since they found an area of degenerative neutrophils surrounding hyphae particles in the skin biopsies [97], findings also observed by Dr. Ogawa and colleagues later on [98]. Recently, our group demonstrated that neutrophils kill conidia and hyphae in vitro in a distinct manner. Whilst conidia killing relies on phagocytosis and ROS production, neutrophils kill hyphae particles through ROS-independent Neutrophil Extracellular Trap (NET) release [47]. These findings, together with the increase of IL-17 in skin lesions of CBM patients, suggest that neutrophil attraction and activation are the most important roles of IL-17 in CBM infection, leading to F. pedrosoi killing and avoiding the spread of the disease.

Another important type of innate immune cell is the DC. These cells are important in the defense against several fungal infections and have a high migratory capacity, cytokine production, and expression of co-stimulatory molecules upon activation, which makes them the perfect bridge between the innate and adaptive immune systems (for a review of DCs and fungal infection, see [99]). Unlike macrophages and neutrophils, the role of DCs in CBM reported in the literature is sometimes contradictory. One of the studies using DCs obtained from the mouse skin (Langerhans cells) showed that the F. pedrosoi conidia block T-lymphocyte activation by inhibiting the CD40 and B7.2 expression in Langerhans cells in vitro [100]. However, our group demonstrated that monocyte-derived dendritic cells from CBM patients positively modulated HLA-DR and CD86 after conidia stimulation in vitro, leading to CD4⁺ Th1 lymphocyte differentiation and proliferation [101]. Since these studies focused on DCs in different parts of the body, more studies are needed to improve the understanding of the DCs’ role in CBM disease.

3.3. Pattern Recognition Receptors (PRRs)

The PRRs–PAMPs interaction is one of the most important and most-studied parts of the host’s strategy in pathogen recognition. The PRRs are located in the cell surface or in intracellular vacuoles, and several pathogen-associated molecular patterns (PAMPS) have been recognized. They are present in large amounts in APCs and innate immune cells, but it can also be found in other immune cells, such as B and T lymphocytes, and also in non-immune cells, such as keratinocytes [102,103], and melanocytes [104], present in our skin. Two of the most studied PRR classes are Toll-like receptors (TLRs) and C-type lectin receptors (CLRs).

Although a variety of studies have demonstrated the importance of these two classes of receptors in different fungal infections [45,105,106,107,108,109,110], little is known about their roles in F. pedrosoi recognition. Recently, a new receptor of DHN-melanin was discovered by Stappers and colleagues and named the Melanin sensing C-type Lectin receptor (MelLec). This discovery might be important for understanding the host immune response in CBM infection, where muriform cells have a higher concentration of melanin in the cell wall. It is the infection morphotype of the fungus, leading to the chronicity of the disease [111] (Figure 1). Another recent study demonstrated that Dectin-1 [112] and Dectin-2 [75] are important in F. pedrosoi recognition, stimulating the Th1 and Th17 responses, respectively. However, CBM agents bind to MINCLE, a new discovery receptor of the CLR family that inhibits and evades the host immune system. Fungal interaction with MINCLE leads to intracellular signaling that blocks IL-12 production (which was stimulated by Dectin-1 activation), inhibiting the Th1 differentiation of CD4+ lymphocytes [113] (Figure 1). This inhibition occurs through the IRF1 degradation after fungal recognition by the MINCLE receptor [112], leading to Th2 differentiation favoring the pathogen survival and infection spread. The MINCLE activation also inhibits Th17 differentiation, but its inhibition mechanism is not clear yet. Recently, Castro and colleagues demonstrated that F. pedrosoi conidia and hyphae activate the NLRP3 inflammasome, an important intracellular receptor belonging to the NOD-like receptor family (Figure 1). NLRP3 activation leads to IL-1β production in DCs and macrophages; however, IL-1β was not essential for the of control CBM disease, since no differences were observed in in vitro and in vivo experiments using NLRP3 KO animals [114]. Although several studies have focused on understanding the role of TLRs in fungal infections, almost no studies can be found in the literature regarding the roles of these receptors in CBM infection. A recent study showed an increase in tlr-2 and tlr-4 gene expression by macrophages infected with F. pedrosoi sclerotic bodies, but not with conidia. Later, our group demonstrated that TLR-2 and TLR-4 are essential receptors for F. pedrosoi conidia recognition and killing by murine neutrophils [47] (Figure 1). Although these receptors are important for conidia phagocytosis and ROS production, they are dispensable for hyphae killing through NET release.

Confirming the in vitro findings, TLR-2KO, TLR-4KO, and Myd88-KO animals infected with F. pedrosoi conidia showed poor prognoses compared with wild-type animals, demonstrating that the TLRs are an essential class of PRRs for the recognition and control of CBM agents [47,113]. Although fungal recognition by TLRs has proved to be important in the early stages of the disease, it seems that F. pedrosoi has the ability to hide from the host’s immune system. Sousa and colleagues (2011) observed that animals pretreated with LPS had a better immune response in CBM, leading to a mild disease followed by its cure. This work demonstrated that F. pedrosoi has the ability to hide or to trick the host immune system by being invisible to the host immune system, leading to the settlement, spread, and chronicity of the disease [113]. This is possible probably due to the cell wall complexity of this fungus, which insufficiently stimulates PRRs, leading to an inappropriate host immune response that favors the fungal survival and disease spread. The authors demonstrated that, after exogenous stimulation of the innate immune system by LPS-TLR4 or imiquimoid-TLR9 activation, the host immune response was restored, providing disease resolution. This costimulatory response also requires the CLR Mincle and signaling via the Syk/CARD9 pathway. LPS is a well-known macromolecule that is able to induce a strong immune response in mammalian cells via the TLR4 pathway, promoting the secretion of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) [115]. Therefore, the treatment of patients with LPS can boost the immune response against fungi and improve their clearance; however, its toxicity impairs its medicinal use [116]. Similar to LPS, imiquimod is a cytokine inducer and an innate immune booster that works through the agonistic activity of TLR7, inducing cell recruitment and activation with IFN-gamma production [117]. Imiquimod is a well-characterized compound. It is FDA-approved and the topical 5% cream is already being manufactured and commercialized [117].

Based on these findings, Souza and colleagues (2014) later reported a new treatment for CBM patients, including a topical cream with the TLR agonist, which was demonstrated to lead to significant improvement of the skin lesion (as discussed below) [118].